The mechanisms by which influenza A virus (IAV) overcomes extracellular biological barriers in the respiratory tract, such as the mucus gel and periciliary layer (PCL), to reach the underlying epithelium and initiate infection remain unclear. Our recent findings suggest efficient penetration of respiratory viruses through the mucus/PCL barrier is critical for infection. The overall objectives in this application are to (i) examine the functional role of virus shape on mucus/PCL penetration and (ii) elucidate the host-associated factors that compromise protective barrier function and physiological clearance mechanisms within the airways that together may provide a permissible environment for IAV infection. The central hypothesis is that susceptibility to IAV infection is enhanced through the combination of shape-dependent penetration of IAV particles to the underlying epithelium and impaired mucus/PCL-facilitated capture and clearance of IAV. The central hypothesis will be tested by pursuing two specific aims: 1) Determine the impact of IAV particle morphology on IAV penetration through the mucus / PCL barrier and 2) Determine the impact of secreted and tethered mucin expression on IAV penetration through the mucus / PCL barrier. In the first aim, we will use high-speed fluorescent video microscopy and multiple particle tracking to compare the penetration of spherical and filamentous IAV through the mucus/PCL barrier of in vitro human airway epithelial (HAE) cultures grown at an air-liquid interface (ALI) that recapitulate the in vivo airway lumen microenvironment. In the second aim, we will use genetically modified HAE-ALI cultures to mechanistically probe the impact of secreted and tethered mucins on IAV penetration, mucus/PCL barrier function, and mucociliary clearance. The research proposed in this application is innovative as it employs a diverse array of tools from biophysics, engineering, and molecular/cell biology to understand how host-pathogen interactions are altered to promote IAV infection. If successful, the results of this work will be significant as such knowledge of IAV-mucus barrier interactions has broad implications for virus transmission, the susceptibility of normal individuals versus those with chronic lung disease to respiratory virus infection, and viral vector design for gene therapy applications.